We have devised site-specific DNA recombination strategies to easily move defined segments of DNA into and out of the mouse genome in order to understand the developmental control of gene expression. In previous work we established that the Cre recombinase of phage P1 can efficiently excise a loxP delimited DNA fragment from the genome of a wide variety of eukaryotes, including mice, and that Cre can also direct integration of loxP-tagged exogenous DNA to a loxP site previously placed into the genome. We have placed recombination substrates at defined chromosomal positions using homologous recombination in embryonic stem (ES) cells. Following Cre-mediated targeting of defined promoter-reporter constructs in these cell lines, mice are being generated that will allow dissection of the regional and temporal specific determinants of gene expression in the proenkephalin promoter. Mechanistic analysis of Cre function should provide an increased ability to direct Cre-meditated events in cultured cells and transgenic mice. We have used mutationally altered lox sites to develop a new Cre-mediated targeting strategy that is 20-100 times more efficient than our previously described strategy in a model cultured cell system. This system is now being adapted to the generation of transgenic mice. We have also identified regions of the Cre protein that allow Cre to localize to the nucleus in eukaryotic cells. This work has led to the development of a novel tool that will be of general utility in the transgenic field: a functional fusion of Cre with Green Fluorescent Protein. The fusion protein allows ready identification of transiently transfected ES cells so that recombinationally committed cells can be easily purified by FACS. We anticipate that this will also be a useul tool for tracing the pattern of expression of Cre expression in transgenic mice. Other tools we have evaluated include an adenovirus expressing Cre that we have used for conditional knockout of the C/EBPa transcription factor gene. These mice show that C/EBPa is required to prevent hyperbillirubinemia, a result that was not anticipated from previous work. To monitor Cre expression in transgenics, we have also developed monoclonal antibodies. To gauge recomibination potential we developed a reporter mouse capable of ubiquitous expression of lacZ after recombination. This latter mouse thus provides a sensitive functional assay for determining the temporal and spatial patten of cre expression and is far superior to conceptually similar mice described by other researchers since it exhibits nearly ubiquitous expression of the reporter gene after activation.